Immune Cell Contribution to Mammary Gland Development

Postpartum breast cancer (PPBC) is a unique subset of breast cancer, accounting for nearly half of the women diagnosed during their postpartum years. Mammary gland involution is widely regarded as being a key orchestrator in the initiation and progression of PPBC due to its unique wound-healing inflammatory signature. Here, we provide dialogue suggestive that lactation may also facilitate neoplastic development as a result of sterile inflammation. Immune cells are involved in all stages of postnatal mammary development. It has been proposed that the functions of these immune cells are partially directed by mammary epithelial cells (MECs) and the cytokines they produce. This suggests that a more niche area of exploration aimed at assessing activation of innate immune pathways within MECs could provide insight into immune cell contributions to the developing mammary gland. Immune cell contribution to pubertal development and mammary gland involution has been extensively studied; however, investigations into pregnancy and lactation remain limited. During pregnancy, the mammary gland undergoes dramatic expansion to prepare for lactation. As a result, MECs are susceptible to replicative stress. During lactation, mitochondria are pushed to capacity to fulfill the high energetic demands of producing milk. This replicative and metabolic stress, if unresolved, can elicit activation of innate immune pathways within differentiating MECs. In this review, we broadly discuss postnatal mammary development and current knowledge of immune cell contribution to each developmental stage, while also emphasizing a more unique area of study that will be beneficial in the discovery of novel therapeutic biomarkers of PPBC.


Introduction
Breast cancer is one of the leading causes of death of women globally as a result of metastatic disease [1].Over the years, there has been a trend in women that have deferred to having their first child after the age of 35, therefore, increasing their risk of breast cancer development [2].This subtype of breast cancer amongst postpartum women, termed postpartum breast cancer (PPBC), is diagnosed within 5-10 years following birth of the last child and is observed in women under 45 years of age [2][3][4][5][6].With this definition, PPBC can be attributed to approximately half of the young mothers to emulate a wound healing signature [20][21][22][23]; however, little effort has been made to confer the 'wound' that prompts this wound-healing environment.Here, we hope to open a dialogue into the consideration of lactation as the inflammatory 'wound' and involution as the wound healing-like modulator of the inflammatory response.The relationship between the inflammatory profile of the mammary gland and neoplastic development is an area of high interest; however, the mechanism of how post-pregnancy stages, specifically lactation, influence PPBC progression requires more rigorous evaluation.Specifically, we suggest that investigating immune pathway activation within differentiating mammary epithelial cells (MECs) will provide more comprehensive knowledge of how the immune system functionally contributes to mammary gland development, as it has been recently proposed that the effector functions of immune cells are partially mediated by MECs and the cytokines they produce [24].
In this review we aim to provide a broad overview of what is currently known of the role of immune cells during the stages of postnatal mammary gland development.We also highlight an area of interest for future studies regarding the exploration of innate immune activation within MECs as a mechanism to uncover potential biomarkers of inflammation that may be beneficial in improving current therapeutic options for young women afflicted with PPBC.In this review we have focused on mouse and human model systems.Studies of postnatal mammary development and PPBC in human subjects are limited due to the sparsity of human breast tissue making mouse models a suitable alternative, as the overall structure of the immune system between the two species is remarkedly similar [2,25].However, it is important to note any significant species differences to prevent assumptions of human immunology that either do not occur or can't be modeled in murine systems [25].

Pubertal Development
The mammary gland is a unique organ that undergoes three stages of development: embryonic development that occurs in utero, as well as pubertal and reproductive development that occur postnatally.Complex interactions between epithelial cells and immune cells within the stroma play an important role in postnatal mammary gland development [20].As MECs differentiate during pregnancy and lactation, these immune cells are exposed to rarely encountered milk proteins that challenge self-tolerance mechanisms [23]; therefore, postnatal development of the mammary gland occurs in an immune-competent microenvironment [20,26].There has been extensive research into how cells of the innate immune system play a role in virgin development.At birth, the mammary gland begins as an epithelial rudimentary tree comprised of multiple layers of luminal 'body' cells that surround the central lumen and corresponding basal cells [27][28][29].Resident macrophages have been found to be in high abundance adjacent to this rudimentary structure at ~ 2 weeks of age [30].In the mouse, postnatal mammary gland development commences with puberty at ~ 3-4 weeks of age [26,31,32].This developmental stage is characterized by ductal elongation and invasion as a result of proliferation of cells throughout the terminal end buds (TEBs).Previous studies have demonstrated that macrophages are responsible for compiling collagen into long structured fibers around TEBs that serve to enhance ductal elongation [20].More recent evidence has determined that macrophages contribute to ductal elongation in a signal transducer and activator of transcription 5 (STAT5)-dependent manner [33].In mammary organoids, it has been established that tumor necrosis factor alpha (TNFα) produced by macrophages activates the PIK3-CDK1/cyclin B1 signaling supporting the activity of mammary stem cells [34].Ductal macrophages have also been determined to respond to tissue damage.Specifically, a multiphoton laser was utilized to generate epithelial cell damage.Following laser damage, 3D subcellular resolution intravital microscopy revealed that ductal macrophages extended dendrites towards the affected areas to facilitate the formation of a secure epithelial-immune interaction [35].Mast cells have also been determined to be necessary for accurate TEB and duct formation, likely via degranulation [20,29,[36][37][38].Eosinophils have also been revealed to be recruited to the stroma area near the head of the proliferating TEBs to facilitate bifurcation [20,30,39,40].This infiltration is a result of augmented expression of the chemokine eotaxin at ~ 5 weeks of age [20].Eosinophil infiltration must be tightly regulated as increased numbers were demonstrated to delay ductal growth [41].Interestingly, decreased numbers of eosinophils resulted in diminished density in mammary gland structures [42,43].Taken together, this data is indicative that a delicate balance of immune cell quantity is required for normal development.
During pubertal development, myoepithelial cells begin laying down extracellular proteins including fibronectin, laminin and type IV collagen that will develop into the basement membrane, which separates the parenchymal and stromal regions [32].The stromal compartment of the mammary gland surrounds the ducts and consists of thick connective tissue, fibroblasts as well as other cell types.Maintenance of ductal structures is contingent upon the health and strength of the basement membrane [31,32].Throughout differentiation, the basement membrane functions to influence epithelial characteristics including shape, growth, polarity and responsiveness to hormones [31,32]; thus, the basement (2024) 29:16 membrane is vital for MEC differentiation and overall function of the mammary gland.At 8-9 weeks of age, the ductal tree fills the entire fat pad [26,[44][45][46].During each estrous cycle, the mammary gland expands in preparation for pregnancy, a time during which alveolar cells commence further differentiation into milk-secreting structures [26,47].If pregnancy does not occur, the gland regresses until the next estrous cycle, which occurs every 4-5 days in mice [29,46,48,49].
Despite the contribution of innate immune cells during virgin development, there is little information regarding adaptive immune cell function throughout mammary gland development [37,39,40,49].A seminal paper from Plaks et al. was the first to shed light on how cells of the adaptive immune response functionally contribute to pubertal development [26].In this study, they observed CD11c + antigen presenting cells (APCs) in close proximity to the mammary epithelium during branching morphogenesis.Their results demonstrated that APCs actively interacted with and proliferated during organoid branching [26].Based on these results, it was hypothesized that T cells would also need to interact closely with the epithelium due to APCs being necessary for proper T cell activation [26].Indeed, they demonstrated that both CD4 + and CD8 + T cells were closely associated within mammary ducts and also co-localized with adjacent APCs [26].A more recent study has identified that CD11c-Cre-mediated depletion of CD11b − CD103 + (DC1) dendritic cells (DCs) in immune regulatory factor 8(IRF8) floxed mice resulted in a small increase in branching within pubertal female mice compared to their control counterparts [35].
The most significant knowledge gained from the work from Plaks et al. came from the assessment of ductal growth in T cell receptor α (TCRα) deficient mice, which lack CD4 + and CD8 + T cells.It was found that there was accelerated ductal outgrowth as well as augmented branching in mammary glands lacking this T cell response [26].These results were the first to demonstrate that CD4 + and CD8 + T cells negatively regulate mammary epithelial development.Additional studies will be required to have a more comprehensive understanding of various T cell subtypes throughout mammary gland development including pregnancy and lactation.Currently, data collected utilizing mouse models suggests that cells of innate and adaptive immunity exert contrasting effects during mammary gland development [50].These results indicate that cells of the innate response serve to enhance pubertal development via secretion of cytokines including TNFα that stimulate proliferation of MECs.On the contrary, adaptive immune cells negatively regulate luminal differentiation via secretion of IFNγ [26,50].

Pregnancy
Pubertal mammary gland development is stimulated via hormonal signals from both the ovaries and pituitary gland; however, the pituitary hormone prolactin, is the most influential during pregnancy [44].At the onset of pregnancy, upregulation of prolactin and progesterone promotes rapid proliferation of MECs, resulting in an increase in both epithelial cell number and surface area [44].MECs also undergo a metabolic transition in which there are alterations to their gene expression profiles to favor impending demands for increases in energy production that are necessary for ensuing lactation [51,52].This period of augmented cell proliferation and metabolic activity generates replication fork stress as well as reactive oxygen species (ROS) that can promote oxidative damage to DNA, lipids and proteins that are required for cellular proliferation [53].Replication stress is generally defined as a slowing or stalling of the replication fork complex [53,54]; however, during pregnancy it is hypothesized to occur as a result of rapid proliferation of MECs [53].During this time, epithelial cells are vulnerable to replication stress that may result in DNA damage if unrepaired.This damaged DNA has the potential to leak into the cytosol resulting in activation of cytosolic DNA sensors such as cyclic GMP-AMP synthase (cGAS).cGAS initiates activation of the stimulator of interferon genes (STING) that ultimately elicits downstream production of type I interferons and proinflammatory cytokines via activation of IRF3 and nuclear factor kappa B (NF-κB), respectively [55][56][57][58].Studies have illustrated that throughout pregnancy, intracellular sensors will prompt activation of cellular stress responses such as those involved in preserving genome integrity, DNA damage repair, and cell cycle progression.Specifically, p53 and BRCA1 have been observed to be upregulated during pregnancy, to suppress tumor development and enhance DNA damage repair, respectively [53,54,59,60].There has also been emerging evidence suggesting crosstalk between pathways associated with genome stability and the immune response [15,55,61].Specifically, innate immune mediators such as the cGAS-STING pathway have been demonstrated to be activated in response to DNA damage and chromosomal instability [62,63].Disruption of these protective mechanisms during pregnancy may predispose replicating MECs to unresolved DNA damage, further encouraging innate immune activation.
In response to hormonal cues at the onset of pregnancy, several regulatory networks are activated prompting alveolar cells to differentiate, resulting in the development of individual lobuloalveoli that are responsible for milk production and secretion during lactation [20, 30-32, 44, 47, 64, 65].By the end of pregnancy, these lobuloalveolar structures will have replaced the adipocytes resulting as T regulatory (T reg ) cytokines [30].T regs secrete inhibitory cytokines such as TGFβ and IL10 to facilitate maintenance of cellular homeostasis and self-tolerance and may contribute to mammary gland development by mitigating the adaptive immune response, specifically, effector T cell functions.This data also illustrated augmented levels of IL4 and IL13 which are hypothesized to directly regulate MEC function in addition to influencing the surrounding immune cell population [71].Interestingly, factors implicated in lineage commitment of luminal epithelial cells during pregnancy are known to also be associated with T cell lineage determination [71].Specifically, a role of STAT6 and its upstream cytokines, IL4 and IL13, has previously been demonstrated in the expansion of the luminal lineage and is also established to reinforce commitment to a type 2 helper (T H 2) T cell lineage [66,71,72].Moreover, MECs in their undifferentiated state secrete type I cytokines, similarly to what has been observed in the polarization and commitment to the T H 1 T cell lineage [71].To date, there are few studies on leukocyte functional involvement during pregnancy and lactation aside from their roles in response to infection.Immune cells elicit vast phenotypic and compositional changes during mammary gland development [50]; thus, studies into the role of immune cells during pregnancy and lactation will be required to enhance our knowledge of leukocyte function under normal developmental conditions.Additionally, further investigations into the immune pathways responsible for the cytokine phenotype of differentiating MECs will delineate the mechanism(s) by which MECs communicate with surrounding immune cells to promote postnatal mammary development [67].
Prolactin is considered the main hormonal driver of mammary gland development during pregnancy and lactation.Several reports have demonstrated that prolactin can stimulate both innate and adaptive immune cells inclusive of natural killer (NK) cells, macrophages, dendritic cells (DCs), neutrophils, T cells and B cells [38,65].Recently, it has been hypothesized that prolactin plays a role in the homing of immune cells into the mammary gland, although the specific mechanism requires further exploration [38,65].Lymphocyte homing is a complex, controlled mechanism that is generally regulated via various factors such as cytokines, chemokines, and addressins [65].It has been reported that prolactin signaling triggers expression of T cell chemokines such as chemokine (C-C motif) ligand 20 (CCL20) and chemokine (C-X-C motif) ligand 9 (CXCL9) in mammary epithelial cells [65,73,74].These chemokines play a role in attracting lymphocytes and DCs and promoting differentiation and expansion of leukocytes to elicit tissue extravasation [65]; therefore, prolactin plays a role in indirect T cell mobilization from adjacent mucosa-associated lymphoid tissues to the mammary gland throughout pregnancy [38, in a functional milk-producing gland.Myoepithelial cells remain uniquely positioned around the alveoli to permit direct contact between epithelial cells and the basement membrane.This interaction is established to be essential for full MEC differentiation and subsequent milk secretion [32].Due to their contractile nature, myoepithelial cells also serve to facilitate the movement of milk during lactation [31,32].Pregnancy denotes the most dramatic expansion of the mammary epithelium and alterations in the surrounding stroma.It has been previously reported that immune cells and their cytokines influence epithelial cell reorganization and differentiation into alveolar structures [20,64,66]; therefore, communication between epithelial and stromal cells is essential for differentiation into a milk-producing gland [22,30,31,34,40,64,67].Studies regarding immune cell function in mammary gland development during pregnancy and lactation remain limited.During pregnancy, macrophages, eosinophils and mast cells have been established to accumulate adjacent to and in alignment with alveolar structures, though their functional contributions are less well-studied [20,30,38].Despite this lack of knowledge, the infiltration of immune cells during pregnancy is hypothesized to contribute immune cells to the milk and effect immune surveillance due to increased risk of inflammation of the breast tissue that may involve infection, commonly referred to as mastitis [22,68,69].There is some evidence suggestive that these cells also have important functional roles in development of the mammary gland during pregnancy.Mice lacking epithelial-associated macrophages exhibited pre-mature and aberrant alveoli formation during pregnancy in a colony stimulating factor 1 (CSF1)-dependent manner [29,70].This data is indicative that macrophages have important functional roles in mammary development, although more rigorous evaluation will be of benefit to further elucidate how other immune cells functionally contribute to pregnancy.
Throughout pregnancy, the mammary epithelium secretes cytokines to regulate lineage commitment and differentiation.In vitro, MECs in their undifferentiated state secrete type I cytokines such as IL12a, IFNγ and TNF [66,71].These cytokines favor subsequent development of a strong cellular immune response via stimulation and activation of both innate and adaptive immune cells such as natural killer (NK) cells, neutrophils, macrophages and T cells.Upon differentiation in the presence of lactogenic hormones such as prolactin and dexamethasone, MECs switch to production and secretion of type II cytokines such as IL4, IL13 and STAT6 [66,71].This type II response directs regulation of inflammation, antibody production as well as effector T cell responses.Transcriptome analysis of total RNA from mammary tissue during pregnancy and lactation exhibited increased expression of immune mediators such 1 3 mammalian development [87].ROS are key signaling molecules and also play a vital role in the progression of inflammatory diseases [84].Release of mitochondrial components into the cytosol has been shown to result in activation of toll-like receptors (TLRs), the cGAS-STING pathway [87][88][89] and the inflammasome [90,91], noting the intimate relationship between mitochondrial homeostasis and innate immune pathways [87-89, 92, 93].Promoting mitophagy during MEC differentiation is required to gain more efficient mitochondria to meet the high energetic demands of lactation.Our lab has recently published that mitophagy is required for MEC differentiation [51].Eliminating damaged mitochondria also aids in mitigating activation of innate immune mediators as MECs endure metabolic stress during lactation.These data suggest that lactation is likely a key driver of innate immune activation and highlights the need for further studies to assess innate immune activation within MECs (Fig. 1).Mitochondrial regulation of the immune system is relatively understudied in the mammary epithelium as past research of immunity during lactation have fixated on the transfer of passive immunity from the mother to the newborn during breastfeeding.
Breastfeeding promotes the postnatal transfer of nutrients and immune factors from the mother to the infant, enhancing protection against both respiratory and gastrointestinal infections in the immunologically naïve newborn [20,94].Immune cells passively transferred from mother to newborn serve to compensate for developmental delays in the infant immune system.This delay is thought to occur because, in utero, the fetus is protected by the maternal immune system from most pathogens and therefore does not require the mature immune system that is necessary for independent survival [94]; thus, nutritional resources and energy can be utilized for the development of essential organ systems that are vital for extrauterine survival [94].Another reason for this delay may also be to circumvent immunologic reactions to maternal tissues in utero.Maternal milk consists of various nutrients, factors and cells that are beneficial upon ingestion by the newborn.Leukocytes are thought to migrate via lymphatic vessels and systemic circulation into the breast where it is believed they enter the lumen of the alveoli via the paracellular pathway [69].Specifically, these immune cells must undergo a transepithelial migration in which they must cross the ductal basement membrane, make their way around the myoepithelial cells and finally transverse the luminal epithelial cell layer to enter the alveolar lumen [30,38].
Previous studies have observed that macrophages and neutrophils are present in breast milk [Wirt 1992].Breast milk leukocytes not only protect the infant but also protect the mother from infection during lactation.Conditions such as trauma, inadequate emptying of the breast, and 65].This process likely involves multifaceted communication between the mammary epithelium and infiltrating T cells in addition to induction of chemokine and adhesion molecule expression.In support of this, recent studies have demonstrated that both macrophages and T cells are not only intimately associated with the mammary epithelium but are in fact embedded within the bilayer, which is ultimately suggestive of a necessity for epithelial-immune interactions [50,67].Interestingly, prolactin is also responsible for the movement of fluid, ions, and immune cells into the milk in preparation for lactation [38].

Lactation
With the onset of lactation, there is a substantial increase in energetic demand as lobualveolar structures produce milk, which serves as a primary nutrient source for the offspring [51,52].During pregnancy, a metabolic shift commences in favor of supporting transport of glucose and amino acids to the developing fetus [75].Once lactation begins, resource allocation adjusts from storage prioritization to augmented milk production in response to increased energy and nutrient demand [75].Heightened energetic demand during lactation creates an enhanced probability of generating ROS which can cause cell damage.Reports have demonstrated that ROS can serve as signaling stimuli, however, a delicate balance between oxidants and antioxidants must be maintained to mitigate cellular damage [75].MECs have been observed to upregulate several mechanisms to modulate cellular stress and damage triggered by lactogenic differentiation [52,60,[76][77][78][79]. Mitochondrial function is vital to energy production; therefore, mitochondrial homeostasis must be maintained in metabolically active tissues such as the lactating mammary gland.Mitophagy, the process of recycling damaged mitochondria, is the primary way in which mitochondrial homeostasis is sustained [51,52].Moreover, this process is a key player in many mitochondrial stress responses.There is emerging evidence that supports a role of mitophagy during cellular differentiation as well as tissue development, a process that has been termed 'programmed mitophagy' [51,80].This process differs from other forms of nutrient or chemically induced mitophagy in that it occurs in response to developmental stimuli.Lactogenic differentiation results in severe metabolic stress [52] that results in damage to mitochondrial proteins, lipids and DNA, a consequence of which includes a loss of mitochondrial membrane integrity and potential [81][82][83].Several lines of evidence suggest that mitochondria contribute to activation of innate immune pathways following cellular damage and stress due to the generation of ROS [84][85][86], and that maintenance of mitochondrial dynamics is vital in 94,95].B cells are main components of adaptive humoral immunity.These cells serve to produce immunoglobulins, such as IgA, that enters the milk to elicit protection against intestinal and respiratory pathogens in the recipient infant [20,94].In support of this, IgA-producing B cells have been observed near alveolar cells so that IgA can be released into the milk [38,39].Moreover, IgG and IgM have also been found in breast milk, although in much smaller concentrations [94,95].Despite the importance of immunoglobulins in breast milk, most lymphocytes in breast milk are T cells, as determined by the presence of CD3 [94,95].It was verified that these T cells exhibited a memory phenotype as observed by expression of CD45RO as well as significantly more phenotypic markers of activation in comparison to T cells within the peripheral blood [69,94,95].Breast milk T cells and macrophages illustrated a more motile phenotype ductal blockage can trigger an inflammatory response in the mammary gland, often resulting in severe mastitis, abscess or septicemia [69].To facilitate in mitigation of these circumstances, there is an increased influx of leukocytes into the mammary gland.Functional alterations in breast milk leukocytes such as neutrophils and macrophages have been discovered in comparison to those present in the peripheral blood [94,95].Following stimulation with chemoattractants, it was observed that breast milk neutrophils and macrophages exhibited increased expression of CD11b as well as decreased expression of L-selectin [94]; therefore, the adherence, orientation and movement of breast milk neutrophils and macrophages were drastically diminished [95].These leukocytes were also shown to exhibit known markers of activation [95].Lymphocytes, inclusive of both B and T cells have also been observed in breast milk [38, 39, 69, Fig. 1 Lactation Challenges Mitochondrial Homeostasis.The lactating mammary gland challenges mitochondrial homeostasis via necessitating maintenance of a substantially demanding differentiated state.The mitochondria are under immense metabolic stress due to increased energetic demands as mammary epithelial cells work to produce milk.This stress results in augmented production of harmful reactive oxygen species (ROS) that result in mitochondrial damage/dysfunction.Under conditions of stress or damage, the mitochondria releases its components into the intracellular space to serve as damage associated molecular patterns (DAMPs).Aberrant mitochondrial components have been shown to elicit activation of innate immune modulators such as Toll-like receptors (TLRs), the inflammasome and the cGAS-STING pathway, all of which result in an inflammatory response.Ultimately, activation of innate immunity in response to mitochondrial damage promotes an environment of sterile inflammation; thus, innate immune activation during lactation is of interest for future studies As described above, a proposed function of lactationassociated immune cells is to provide immune surveillance and protection against possible infection.Until recently, bacterial infection has been the alleged cause of mastitis; however, there is accumulating evidence that infection may be an indirect consequence of an unidentified primary cause of the disease [11,105].The World Health Organization recognizes milk stasis as an indirect origin of mastitis [106].The build-up of milk within the breast increases intraductal pressure triggering sterile inflammation that may generally be followed by a secondary bacterial infection [11].The theory that sterile inflammation could be a primary cause of mastitis is not a new concept [107], however, the scarcity of breast tissue from women with mastitis creates challenges in further investigations of sterile inflammation being a causal agent.It has also been illustrated that innate immune receptors recognize intra-and extracellular molecules such as aberrant DNA and cellular components released as a consequence of breast engorgement, ultimately resulting in a potent innate immune response and milk stasis [105,108].This evidence further supports that lactation has an inflammatory profile that is of interest for future studies regarding PPBC initiation.
Recently, work from Oakes et al. demonstrated that mutation in the oligoadenylate synthase 2 (OAS2), a sensor of viral dsRNA, resulted in postpartum failure of lactation in mice that exhibited normal mammary gland development [105].They observed drastically diminished synthesis of milk proteins in addition to inhibition of proliferation, cell death and a robust interferon response.These results were also reiterated in vitro using HC11 and T47D mammary cells [105].These studies established, for the first time, that stimulation of an innate immune pathway in MECs can elicit milk stasis, therefore, introducing a novel therapeutic target for anti-inflammatory therapy [105].Further investigations of immune activation within MECs may facilitate our understanding of postpartum mammary gland development, ultimately improving postpartum breast health and decreasing instances of PPBC [67].Lactation represents a time of metabolic stress that ultimately promotes sterile inflammation that challenges cellular homeostasis.During times of stress, cells either adapt or die [52].Recent data has demonstrated decreased antigen-dependent T cell expansion during lactation that is predicted to assure successful lactation by increasing self-tolerance mechanisms to milk [23].Taken together, this evidence maintains that there is a delicate balance of immune activation/suppression within the lactating mammary gland to encourage MEC survival during functional mammary gland development.
Current evidence leads us to anticipate that cancer cells utilize intrinsic mechanisms of immune suppression during lactation to evade immune surveillance and promote the compared to their blood counterparts, which is indicative of selective migration of these cells from the circulation into the milk [69].Leukocytes within the milk have been observed to share morphologic characteristics similar to epithelial cells including the presence of lipid droplets, cytoplasmic vacuolation, as well as an enlarged nucleus or multinucleation [69].Several pattern recognition receptors (PRRs), such as TLRs, that function to recognize molecular patterns of pathogens, have also been discovered in breast milk [96].Interestingly, it has been hypothesized that lactation evolved as an inflammatory response to tissue damage and infection following birth, and that specific inflammatory molecules have become fundamental modulators of lactation [96].We predict that exploration into immune activation within MECs will uncover inflammatory modulators of lactation.In support of this, past investigations have demonstrated that prolactin is not only a vital lactogenic hormone but can also serve as an anti-inflammatory or proinflammatory cytokine depending on the cellular milieu [96,97].This evidence implies that lactation exhibits a delicate inflammatory balance.Moreover, transcriptional single cell analysis using human milk suggests that essential cellular dynamics will enhance understanding of the association of lactation with breast cancer development [98].
During lactation, the mammary epithelium must become tolerogenic to milk components [22,23,99] and adopt a partially suppressive immune environment to permit tolerance to self-antigens [23,100].There is evidence that various immune cell types are present during lactation, although the mechanism(s) directing their functional contributions remain limited [29,49,[101][102][103].Work from Wilson et al. has demonstrated that chemokine receptors are responsible for directing macrophage dynamics during normal development [102].On the other hand, a study from Cansever et al. demonstrated that mice deficient in lactation-induced macrophages did not exhibit alterations in milk protein content, or structural variations.This data is suggestive that macrophages are not essential to the foundation of tissue integrity within the lactating mammary gland [103] and may function to grant immune surveillance and enter the milk to facilitate passive immunity transfer to the newborn.Additional studies regarding the type and distribution of macrophage populations are reviewed elsewhere [101,104].Interestingly, progeny from IL5 deficient mice, that have diminished numbers of eosinophils, were observed to be underweight and also exhibited high preweaning mortality rates [42].Taken together, this data is indicative that further investigations into all immune cell types will provide insight into their regulation and effector functions to provide a more comprehensive understanding of how the immune system influences lactation. of macrophages function in tissue remodeling, immunosuppression and wound repair [20].Recent studies have uncovered that tissue resident macrophages are uniquely confined to the basal-luminal interface during involution as a superior position for surveillance of damaged and/or dying cells [120].Ablation of mammary ductal tissue-resident macrophages achieved via either anti-CSF1R or CD11c-DTR (diphtheria toxin receptor) uncovered that other immune cell populations had diminished capabilities in the clearance of alveolar cells during involution [35].Moreover, shortterm depletion of CD11c + cells with use of CD11c-DTR revealed delayed remodeling as determined by the persistence of sizeable alveolar lumens [35].This was determined to be a result of a loss of CC3 + cell phagocytosis, noting the vital role of ductal macrophages in clearing cellular debris [119].In addition to macrophages, eosinophils and mast cells have been observed to contribute to regeneration of adipocytes and ECM restructuring [40,122,123].A unique subtype of NK T-cell like (NKT) cells have also recently been observed during late-stage involution [67].These cells differ from typical NKT cells in that they express γδ T cell receptors (TCRs) instead of αβTCRs, indicating a role in specialized antigen recognition that permits inhibition of mammary oncogenesis [67].Many of the infiltrating immune cells throughout mammary gland development are involved in wound healing [20]; therefore, enhancement of knowledge on the process of wound healing as well as the insulting agent triggering the inflammatory response will be beneficial for understanding immune cell function throughout postnatal mammary development.
Regulation of immune cell infiltration, activation and differentiation in the involuting mammary gland remains unknown.Previous data has suggested that involution is associated with an influx of neutrophils followed by macrophages and lymphocytes [68].Effector T and B cells have been observed within the involuting mammary gland via the presence of CD4 + , CD8 + , and CD19 + cells, respectively [22].These lymphocytes are largely believed to enhance defense against infection such as mastitis in the mother and respiratory and gastrointestinal infections in the newborn.Neutrophils are also critical in preventing mastitis as they function to eradicate bacteria through phagocytosis [68].More recent studies are revealing that, in the mouse, epithelial cells are responsible for the clearance of most dead cells and residual milk proteins [20,124].Specifically, there has been evidence suggestive that MECs are capable of functioning as non-professional phagocytes to facilitate neutrophils and macrophages in the clearance of milk and cellular debris [20,124].In vitro, upon exposure to dead cells, MECs exhibited cytoskeletal actin reorganization, membrane ruffling and micropinocytosis [124].Epithelial cells, endothelial cells and fibroblasts have been observed development of a pro-tumorigenic microenvironment [109][110][111][112]. Increased expression of the inhibitory programmed death-ligand 1 (PDL1) in triple negative breast cancer reinforces this theory [113,114].Tumor cells express PDL1 which binds to its receptor, programmed death-1 (PD1), on T cells to inhibit their effector functions; thus, diminishing the adaptive immune response.Moreover, cancer cells have also demonstrated use of other normal cellular pathways such as those related to angiogenesis and sustained proliferation to facilitate their survival [115].Several risk factors for cancer are associated with inflammatory mechanisms [13,[16][17][18], thus, the lactating mammary gland is becoming a conceivable environment to facilitate the promotion of PPBC development.Knowledge regarding the exact mechanisms of immune regulation during lactation remain limited; therefore, further exploration is required to elucidate how neoplastic cells utilize this time of inflammation to evade detection.We anticipate that investigations into this research area will uncover novel biomarkers for PPBC development, resulting in improved treatment options for women.

Involution
Following lactation, the mammary gland undergoes involution resulting in a structure similar to that of the mature virgin gland [20].Involution is categorized into two distinct phases: an acute, reversible phase followed by an irreversible phase.The acute phase is characterized by diminished synthesis and secretion of milk, in addition to STAT3dependent lysosomal-mediated programmed cell death [29,51,[116][117][118].In the mouse, this initial phase typically occurs 1-3 days after weaning and is reversible.Generally, 72 h after weaning the irreversible phase begins.This phase is described as continued epithelial cell death, immune cell infiltration and tissue remodeling [21,23,35,[118][119][120][121].This is followed by differentiation and repopulation of the stromal microenvironment by adipocytes [68].CXCL1, a neutrophil chemoattractant, has been observed to be induced on day 1 of involution [20,22].By days 3-4, genes associated with macrophage chemoattraction, and differentiation were observed to be upregulated [22].These include CCL6, CCL7, CCL8, and CXCL14 followed by macrophagespecific antigens such as CSF1 receptor (CSF1R), CD68 and low-density lipoprotein-related protein (LRP1), all of which exhibited peak expression 72-96 h post weaning [22].In response to alterations in the surrounding stroma, monocytes mature into various macrophage phenotypes.During involution, monocytes specifically differentiate into M2 macrophages that are alternatively activated via cytokines such as IL4, IL10 and IL13 [20,22].This subtype as a 'wound' that prompts the wound-healing signature during involution.Effector functions of immune cells within the developing mammary gland have been suggested to be influenced by MECs and they cytokines they produce.While functional contribution of immune cells is an area of high interest, we also propose that investigations into activation of innate immune pathways within MECs will be extremely beneficial in accessing how the immune system contributes to normal mammary development.We believe that future investigations aimed at accessing how lactation contributes to PPBC will facilitate the discovery of novel inflammatory biomarkers that can be targeted using immunotherapeutic treatment.Particularly, STING agonists have gained interest in breast cancer therapeutics and have shown promise in enhancing the efficacy of current immunotherapies [139][140][141][142]. Women diagnosed with PPBC have poorer prognoses and we believe that expanding our depth of knowledge regarding post-pregnancy developmental stages, particularly lactation, will facilitate the development for improved treatment options and better patient outcomes.
to function as amateur phagocytes to facilitate the removal of neighboring dead cells via phagocytosis [68].Clearance of dead cells is crucial as it helps avert inflammation resulting from cell lysis through stimulating the secretion of anti-inflammatory cytokines [64,71].Dead cells that are not phagocytosed lose contact with the basement membrane and are released into the ductal lumen where they are accessible to professional phagocytes for disposal [68].
Mammary gland involution has been identified as a tumor promoting environment that contributes to the development and progression of PPBC [10,22,125].Involution is known to exhibit wound healing inflammation and an immunosuppressive phenotype, both of which are hallmarks of cancer associated with tumor promotion and progression [23,119,120,[126][127][128][129].This unique phenotype during involution likely occurs to support tolerance in a state of massive cell death.As previously described, STAT3 is an important mediator of programmed cell death within the involuting mammary gland.Importantly, STAT3 is also a key orchestrator of immune suppression [130] and has been revealed to be essential in promoting an immunosuppressive microenvironment when expressed in epithelial cells, ultimately, supporting tumor initiation and progression [131].Additional mechanisms of how involution facilitates the development and progression of PPBC have been reviewed in further detail elsewhere [10,22,120,125,[132][133][134][135].

Conclusions
In conclusion, the mammary gland serves as a unique model to study how the immune system contributes to postnatal tissue development.Extensive work has been published on pubertal development and the process of involution, although mechanisms regarding immune cell functions that contribute to normal mammary development during pregnancy and lactation remain largely unexplored.In addition to the mammary gland, other tissues, including the brain and liver continue to develop postnatally.Emerging evidence has demonstrated that cytokines secreted by CD4 + T cells in the brain indirectly enhance spatial learning and memory as well as influence cell survival, proliferation and differentiation [136].In the liver, a recent study demonstrated that signaling from macrophages resulted in recruitment of T regs and thus, immunosuppressive activity during postnatal development [137].In the breast, recent data is suggestive that pregnancy and lactation may permanently alter certain aspects of the molecular histology of the mammary gland, resulting in an increased risk for postpartum breast cancer development [4-6, 125, 138].Involution is widely accepted as a driver of PPBC development due to its wound-healing attributes.Here, we suggest the consideration of lactation